Project Title: „Obtaining and characterization of virus-like particles of novel RNA phages ”

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/16/A/104

Period: 1 April 2017 – 31 March 2020

Project costs: 648 640, 00 EUR

Principle Investigator: Dr. biol. K. Tārs

Project summary:

Virus-like particles (VLPs) are non-infectious protein shells assembled from viral coat proteins (CPs) with a variety of applications in both fundamental and applied research. VLPs of ssRNA phages have been widely utilized for creation of new vaccines, as nanocontainers for targeted drug delivery and for bio-imaging purposes, but the currently available range of ssRNA CPs is too limited for further developments in the field. During the project, we aim to produce VLPs from more than 50 new ssRNA phage CPs that have been recently identified in metagenomic datasets. Depending on the properties of the novel VLPs, including expression level, thermal stability and sequence variations of the CPs, we plan to select at least five candidates for thorough characterization, which will include development of effective VLP production and purification protocols, determination of the three-dimensional structure of the VLPs and exploration of VLP tolerance to chemical and genetic modification.

Information published 03.04.2017.

Progress  of the project

1 April 2017 – 30 June 2017

Until 30.06.2017 the following has been achieved:

• 40 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 2 constructs; EM pictures of VLPs for both constructs obtained
• 2 VLPs obtained and characterized
• 1 VLP crystallized
• Structure determination of 1 VLP in progress

Information published 30.06.2017.

Progress  of the project

1 July 2017 – 30 September 2017

Until 30.09.2017 the following has been achieved:

• 60 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 60 constructs; EM pictures of 30 VLPs obtained
• 30 VLPs obtained and characterized
• 2 VLPs crystallized
• Crystal diffraction data collected from 2 VLPs
• Structure of 1 VLP determined

Information published 29.09.2017.

Progress  of the project

1 October 2017 – 31 December 2017

Until 31.12.2017 the following has been achieved:

• 65 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 65 constructs; EM pictures of 37 VLPs obtained
• 37 VLPs obtained and characterized
• 10 VLPs obtained in preparative amounts
• 5 VLPs crystallized
• Crystal diffraction data collected from 3 VLPs
• Three-dimensional structure of 2 VLPs determined

Information published 02.01.2018.

Progress of the project

1 January 2018 – 31 March 2018

• 80 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 80 constructs; EM pictures of 50 VLPs obtained
• 50 VLPs obtained and characterized
• 12 VLPs obtained in preparative amounts
• 5 VLPs crystallized
• Crystal diffraction data collected from 4 VLPs
• Three-dimensional structure determined for 3 VLPs
• Three-dimensional structure of 3 VLPs determined
• For 5 VLP possibility to attach foreign epitopes by genetic fusion checked

Information published 29.03.2018.

Progress of the project

1 April 2018 – 30 June 2018

• 100 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 100 constructs; EM pictures of 70 VLPs obtained
• 70 VLPs obtained and characterized
• 20 VLPs obtained in preparative amounts
• 10 VLPs crystallized
• Crystal diffraction data collected from 5 VLPs
• Three-dimensional structure determined for 5 VLPs
• For 10 VLPs possibility to attach foreign epitopes by genetic fusion checked

Information published: 29.06.2018.

Progress of the project:

1 July 2018 – 30 September 2018

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 75 VLPs obtained
• 75 VLPs obtained and characterized
• 30 VLPs obtained in preparative amounts
• 12 VLPs crystallized
• Crystal diffraction data collected from 6 VLPs
• Three-dimensional structure determined for 6 VLPs
• For 12 VLPs possibility to attach foreign epitopes by genetic fusion checked

Information published 28.09.2018.

Progress of the project:

1 October 2018 – 31 December 2018

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 75 VLPs obtained
• 75 VLPs obtained and characterized
• 40 VLPs obtained in preparative amounts
• 15 VLPs crystallized
• Crystal diffraction data collected from 10 VLPs
• Three-dimensional structure determined for 8 VLPs
• For 20 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 10 VLPs possibility to attach foreign epitopes by chemical coupling checked

Information published 28.12.2018.

Progress of the project:

1 January 2019 – 31 March 2019

Until 31.03.2019 the following has been achieved:

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 75 VLPs obtained
• 75 VLPs obtained and characterized
• 55 VLPs obtained in preparative amounts
• 17 VLPs crystallized
• Crystal diffraction data collected from 12 VLPs
• Three-dimensional structure determined for 10 VLPs
• For 25 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 15 VLPs possibility to attach foreign epitopes by chemical coupling checked
• Article about VLP production and characterization sent in for publication

Information published 29.03.2019.

Progress of the project:

1 April 2019 – 30 June 2019

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 80 VLPs obtained
• 80 VLPs obtained and characterized
• 60 VLPs obtained in preparative amounts
• 20 VLPs crystallized
• Crystal diffraction data collected from 15 VLPs
• Three-dimensional structure determined for 12 VLPs
• For 35 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 20 VLPs possibility to attach foreign epitopes by chemical coupling checked
• Article about VLP production and characterization published in “Journal of Nanobiotechnology” (IF=5.29)

Information published 28.06.2019.

Progress of the project:

1 July 2019 – 30 September 2019

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 80 VLPs obtained
• 80 VLPs obtained and characterized
• 60 VLPs obtained in preparative amounts
• 28 VLPs crystallized
• Crystal diffraction data collected from 15 VLPs
• Three-dimensional structure determined for 15 VLPs
• For 37 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 22 VLPs possibility to attach foreign epitopes by chemical coupling checked
• Article about VLP production and characterization published in “Journal of Nanobiotechnology” (IF=5.29): https://www.ncbi.nlm.nih.gov/pubmed/31084612

Information published 30.09.2019.

Progress of the project:

1 October 2019 – 31 December 2019

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 80 VLPs obtained
• 80 VLPs obtained and characterized
• 60 VLPs obtained in preparative amounts
• 28 VLPs crystallized
• Crystal diffraction data collected from 22 VLPs
• Three-dimensional structure determined for 22 VLPs
• For 40 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 25 VLPs possibility to attach foreign epitopes by chemical coupling checked
• Two publication manuscripts about three-dimensional structures of VLPs and about VLP modification are under preparation

Information published 30.12.2019.

Progress of the project:

1 January 2020 – 31 March 2020

• 116 genes of phage coat proteins (CPs) in expression plasmids obtained
• Expression level and solubility checked for 116 constructs; EM pictures of 80 VLPs obtained
• 80 VLPs obtained and characterized
• 60 VLPs obtained in preparative amounts
• 28 VLPs crystallized
• Crystal diffraction data collected from 22 VLPs
• Three-dimensional structure determined for 22 VLPs
• For 40 VLPs possibility to attach foreign epitopes by genetic fusion checked
• For 62 VLPs possibility to attach foreign epitopes by chemical coupling checked
• Two publication manuscripts about three-dimensional structures of VLPs and about VLP modification have been prepared and submitted to journals

Information published 31.03.2020.